Mastitis in dairy cows is the potentially fatal persistent inflammation of the udder tissue. Mastitis can be caused by numerous bacteria (e.g. staphylococcus and streptococcus strains, E. coli strains, and others). Mastitis has huge implications for herd health, quality and quantity of milk production, and the shelf life of (pasteurized) milk products. It is absolutely essential that mastitis is continuously monitored and managed to minimize its huge economic impact, which is estimated to be more than 2 billion dollars per year in the US. The big economic impact of mastitis is the result of several factors, such as temporary or permanent loss of milk production (this includes discarded milk after antibiotic treatment), reduction in milk price due to poor milk quality, lower value of culled cattle meat, and increased costs for veterinary care and labor for cattle husbandry and preventive measures. In order to avoid substantial financial losses, mastitis has to be detected and treated at the subclinical stage. However, established tests for mastitis that can be performed in real time and on the dairy farm, such as the California Mastitis test (CMT), pH-measurements, electrical conductivity tests, or on-site somatic cell counts, are only indicative, but not conclusive of the infection status of the animal.
Approx. 15 percent of all dairy cattle show signs of mastitis during each lactation period. Conservative estimations attribute the economic impact of mastitis due to lower amounts of produced milk, culling and replacement of severely infected cattle, as well as treatment of mastitis, to approx. 10 percent of the value of milk that is generated (between 50 and 60 million dollars per year in Kansas). It is anticipated that the losses caused by mastitis can be cut at least by 50-70% when mastitis is detected in the sub-clinical instead of the clinical stage. Therefore, there remains a significant need in the industry for technologies for subclinical detection of mastitis.
In addition to mastitis concerns, there are other needs in the industry related to monitoring the quality of milk production. The growth of (usually gram negative) psychrotrophic bacteria is not impeded under conditions where milk is stored (<7° C.). Commonly found psychrotrophic bacteria are species of Pseudomonas, Flavovacterium, Alcaligenes, and Acinetobacter, and others. Psychrotrophic bacteria in raw milk produce heat stable proteolytic and lipolytic enzymes, which are not deactivated by pasteurization. These bacteria usually don't cause mastitis. However, they contaminate the milk because they can survive harsh conditions, making the sterilization of dairy equipment difficult. The nanoplatform technology is adapted herein to detect signature enzymes from psychrotrophic bacteria. The main advantage of using this technology is that it is highly sensitive and allows detecting the presence of psychrotrophic bacteria before they are able to spoil the milk and/or drastically reduce the shelf life of pasteurized milk products. Lipolytic enzymes (also called lipases) release fatty acids from milk fat. Milk contains a high amount of fatty acids with short chains (C4: 11 mol %, C6: 5 mol %). Their release causes the milk to taste rancid. The activity of bacterial lipases indicates the presence of psychrotrophic bacteria in raw and pasteurized milk. Therefore, this technology can be used to assess milk quality and predetermine the shelf life of milk products. There are virtually no other methods to predict the limitations of the shelf life of milk products due to the activity of psychrotrophic bacteria, except bacterial cultures, which take several days. The major problem with this approach is that the enzymes from psychrotrophic bacteria (proteases and lipases) remain active after pasteurization, whereas the bacteria, or at least some of the psychrotrophic organisms in milk, don't survive the procedure. Principally, proteases and lipases from psychrotrophic bacteria could be detected by means of immunoassays. This process would take at least a day, and would be significantly more expensive (factor of 10-25) than the approach described here. The major disadvantage of immunoassays is that they are unable to differentiate between active and deactivated enzymes, whereas the inventive assays are specific to active enzymes.